1. Field of the Invention
The present invention relates to molecules that interact with proteins regulating the nuclear hormone receptor family of transcription factors.
2. Description of the Related Art
All of the cells of the lymphohematopoietic system can be generated from a single cell (Nowell et al, 1969; Spangrude et al, 1988; Capel et al, 1990; Jordan et al, 1990; Wu et al, 1968). This pluripotential hematopoietic stem cell (PHSC) is separated from the mature cells of the peripheral blood by a series of intermediate cells of increasingly restricted developmental potential (Hodgson et al, 1979; Magli et al, 1982; Eaves et al, 1992; Jones et al, 1990; Lansdorp et al, 1990). The most restricted are committed to a single lineage and have limited capacity for self-renewal. Recombinant growth factors and clonal culture systems have made it possible to identify and isolate some committed progenitors, but the more immature PHSC has proven more elusive. Neither the exact number of PHSC nor the process by which their number is maintained is known and human PHSCs have not been unambiguously identified (Orlic et al, 1994). Most human hematopoietic progenitors express the CD34 antigen on their surface and this marker has been used extensively to identify and isolate hematopoietic progenitors (Krause et al, 1996). A CD34+CD38− fraction, which constitutes 0.1% of freshly isolated bone marrow cells, contains most of the PHSC activity in normal marrow (Terstappen et al, 1991). PHSC are not undifferentiated cells, but are uniquely specialized cells, whose role is to provide progenitors for the various hematopoietic lineages in a demand-responsive manner, while protecting the stem cell pool from depletion. The molecular basis for this ability is not known but is under intense scrutiny.
Several approaches have been used to try to identify the genes whose products regulate the stem cell pool. Yang et al (1996) made an EST database for CD34+ cells by single pass sequencing of 402 clones from a directional library. Thirty-five percent of the sequences were from previously unknown genes but none of these were differentially expressed in PHSC. Graf et al (1995), using differential display to identify differences between CD38HI and CD38LO cells, identified one previously unidentified sequence (“345”), that was expressed at ˜2.5 times higher concentration in the CD38LO population. The sequence contained no open reading frame and lacked a polyadenylation site. The pace of the search for hematopoietically relevant genes has quickened lately. Using a cDNA library prepared from CD34+ cord blood cells, close to 10,000 ESTs were identified (Mao et al, 1998). The majority of these were either known sequences (47.6%) or corresponded to previously catalogued ESTs (26.4%), but 14.3% were new ESTs. A retroviral gene trap vector that selects for integration in or near expressed 5′ exons has also been used in an attempt to identify genes that were repressed during hematopoietic differentiation (Muth et al, 1998). Two genes, of unknown function, were identified but targeted deletions failed to show hematopoietic abnormalities.
In all living creatures, cells are continuously dying. They are either killed by injurious agents or they are induced to commit suicide. Cells that are damaged by injury, such as by mechanical damage, exposure to toxic chemicals, undergo a characteristic series of changes: they swell (because the ability of the plasma membrane to control the passage of ions and water is disrupted) and the cell contents leak out, eliciting an inflammatory response in the surrounding tissues. Cells that are induced to commit suicide shrink, have their mitochondria break down with the release of cytochrome c, develop bubble-like blebs on their surface and undergo DNA and chromatin fragmentation. Ultimately they break into small, membrane-wrapped, fragments which are engulfed by nearby phagocytic cells without causing inflammation. The pattern of events in death by suicide is called programmed cell death or apoptosis.
Programmed cell death is needed for remodeling of structures during development. Well-known examples include the resorption of the tadpole tail at the time of its metamorphosis into a frog; the formation of the fingers and toes of the fetus by removing (by apoptosis) the tissue between them, and the sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation.
Programmed cell death is also required to destroy cells that represent a threat to the integrity of the organism. In the immune system, some cell-mediated killing of virus-infected cells occurs by inducing apoptosis and, as cell-mediated immune responses wane, the effector cells are removed by an apoptotic mechanism. Defects in the apoptotic machinery are associated with autoimmune diseases, such as lupus erythematosus and rheumatoid arthritis.
Genetic damage can cause somatic cells to become malignant and lead to abnormal embryonic development (leading to birth defects). Cells respond to DNA damage by increasing their production of p53 which can induce apoptosis. Mutations in the p53 gene, producing a defective protein which does not induce apoptosis, are often found in cancer cells.
The decision to commit suicide depends on the balance between the positive signals needed for survival and signals initiating a death pathway (negative signals). The survival of many cells requires that they receive continuous stimulation from other cells and, for many, continued adhesion to the surface on which they are growing. In the absence of these positive signals, cells initiate a program leading to cell death. Cellular damage by increased levels of oxidants, damage to DNA by these oxidants or other agents like ultraviolet light, X-rays and some chemotherapeutic drugs, as well as agents that bind to specific receptors on the cell surface, also initiate the apoptosis program. These death activators include tumor necrosis factor (TNF)and lymphotoxin that both bind the TNF receptor; and Fas ligand (FasL), a molecule that binds to Fas (CD95).
Cells appear to use different pathways to initiate apoptosis depending on the source of the signal. In a healthy cell, the outer membranes of mitochondria, the endoplasmic reticulum (ER) and the nuclear envelope express the protein Bcl-2 on their surface. Bcl-2 binds a molecule of Apaf-1, which is itself bound to a molecule of caspase 9. Caspase 9 is one of a family of over a dozen caspases (cysteine-aspartate proteases). Internal damage in the cell results in release of cytochrome c from the mitochondria and causes Bcl-2 to release the heterodimer of Apaf-1 and caspase 9. These aggregate in the cytosol. Caspase 9 cleaves and, in so doing, activates other caspases. The sequential activation of one caspase by another creates an expanding cascade of proteolytic activity that leads to digestion of structural proteins in the cytoplasm degradation of chromosomal DNA and death of the cell.
Apoptosis can also be triggered by external signals. Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell. Binding of the complementary death activator (FasL and TNF, respectively) transmits a signal to the cytoplasm that leads to activation of caspase 8. Caspase 8 (like caspase 9) initiates a cascade of caspase activation leading to cell death.
Several known proteins or families of proteins that function to regulate apoptosis include:
1. Members of the BCL-2 family. Some members of this family inhibit apoptosis, including Bcl-2 and Bcl-x. Others, such as BAX, stimulate apoptosis. BAX is thought to accomplish this by binding to and inhibiting the anti-apoptotic functions of Bcl-2 and Bcl-x.
2. FLAMES 1 and 2 are proteins that regulate cell death mediated by receptors of the TNF receptor family. TRAIL receptors are cell death receptors which are members of the TNF receptor family and exert cell suicide effects on cancerous but not normal cells.
3. IAP family members are homologous to the baculovirus IAPs. The open reading frames (ORFs) possess three baculoviral inhibition of apoptosis protein repeat (BIR) domains and a carboxy-terminal RING zinc-finger. The human IAP genes have a distinct but overlapping pattern of expression in fetal and adult tissues. These proteins significantly increase the number of known apoptotic suppressors. A fourth member of the family, termed survivin has also been identified. All appear to be caspase inhibitors.
IEX-IL is an nf-kB regulated survival factor, that protects cells from Fas or TNF induced apoptosis.
The nuclear hormone receptor (NHR) superfamily is a large family of mainly ligand-dependent transcription factors that play a role in the regulation of reproduction, growth, differentiation, and homeostasis. The family includes receptors for steroid hormones (estrogen, androgen, adrenal glucocorticoid, aldosterone, and progesterone), thyroid hormone and retinoic acid as well as a group of so-called “orphan receptors” that have poorly, defined ligand(s).
Members of the family share several structural features including a conserved DNA binding domain (DBD) that targets the receptor to specific DNA sequences which are called hormone response elements (HRE). The carboxyl-end of the receptor contains the ligand-binding domain (LBD) and embedded within this LBD is a hormone-dependent transcriptional activation domain. The LBD serves as a molecular switch that recruits co-activator or co-repressor proteins that regulate transcription of the target genes. Ligand-dependent receptors like the thyroid hormone receptor (T3R) and retinoic acid receptor (RAR) stimulate transcription when ligand is bound and repress it when the ligand is absent (Hu et al, 2000).
The best-characterized mammalian co-repressors are N-CoR (nuclear receptor co-repressor) (Chen et al, 1995) and SMRT (silencing mediator of retinoid and thyroid receptor) (Horlein et al, 1995). These co-repressors fill overlapping but non-redundant roles in regulating transcription. Both SMRT and N-CoR are large proteins (>170 kD) that exist in multi-protein complexes that have an estimated size of 1.5–2 mDa. A SMRT complex, isolated by a combination of conventional and immunoaffinity chromatography has been shown to contain histone deacetylase 3 (HDAC3) and transducin (beta)-like I (TBL1), a WD-40 repeat-containing protein (see below). The HDAC3-containing, SMRT and N-CoR complexes can bind to unliganded thyroid hormone receptors (T3Rs) in vitro (Li et al, 2000). Although both co-repressors are expressed widely, extensive hematological abnormalities including blocks in erythrocyte and T-cell development (Jepsen et al, 2000) follow targeted deletion of N-CoR.
Co-repressors mediate transcriptional silencing by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes (Hu et al, 2000; Li et al, 2000; Wong et al, 1998; Burke et al, 2000). Histone deacetylation, which produces a more compact chromatin structure that is inaccessible to transcriptional activators (Burke et al, 2000), appears to be the predominant means of chromatin modification. Studies of PAR and T3R show that ligand binding leads to the displacement of an HDAC-containing complex from the nuclear receptor in exchange for a histone acetyltransferase (HAT)-containing complex and this may serve as a general mechanism for switching nuclear receptors from a transcriptionally repressive to a transcriptionally active state (Xu et al, 1999). Transcriptional repression by N-CoR involves a co-repressor complex that contains one or more HDAC and may include mSin3A/B (Huang et al, 2000). Changes in repression correlate with alterations in the level of N-CoR and/or SMRT. These levels are regulated by both the rate of synthesis of the co-repressors and, more dramatically, by their rate of degradation. Targeted proteolysis of transcriptional co-regulators has been established as a mechanism for cell-specific regulation of gene transcription (Zhang et al, 1998a). Although the composition of the repressor complex is not fully understood a protein called TBL1 is present in some cells (Huang et al, 2000; Guenther et al, 1998) and in these cells, the extent of transcriptional repression was regulated by the amount of TBL1 present.
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